CN109486685B - Mangrove cuspid and sea lotus endophytic fungus and application thereof in preparation of anti-insect active terpenoid crystal compound - Google Patents
Mangrove cuspid and sea lotus endophytic fungus and application thereof in preparation of anti-insect active terpenoid crystal compound Download PDFInfo
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Abstract
The invention relates to mangrove cuspidate and lotus endophytic fungi and application thereof in preparing an insect-resistant active terpenoid crystal compound, wherein the preservation information of the strains is as follows: the name of the depository: china general microbiological culture Collection center; the address of the depository: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 11/9/2018; the preservation number is: CGMCC No. 16499; and (3) classification and naming: penicillium sp.
Description
Technical Field
The invention belongs to the field of secondary metabolites of mangrove endophytic fungi, and particularly relates to mangrove cuspidate and lotus endophytic fungi and application thereof in preparation of an insect-resistant active terpenoid crystal compound.
Background
The marine-derived endophytic fungi have one of important sources of compounds with novel structure and unique activity. In recent years, some new active compounds have been isolated from the fungus Penicillum, for example: an anti-inflammatory active compound, chrysogenaster, alpha-glucosidase inhibiting compounds chrysines B and C, cytotoxic active compounds penicimenolides B-D and penitalarins A-C, antifungal compound brocapyrrozinA. Early researches find that the crude extract of endophytic fungus Penicillium ethyl acetate has certain anti-insect activity.
Disclosure of Invention
The invention provides mangrove cuspid and lotus endophytic fungus TGM112, which is characterized in that the strain preservation information is as follows: the name of the depository: china general microbiological culture Collection center; the address of the depository: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 11/9/2018; the preservation number is: CGMCC No. 16499; and (3) classification and naming: penicillium sp.
Another embodiment of the invention provides the application of the mangrove cuspidate and sea lotus endophytic fungus TGM112 in the preparation of mixed source terpenoids 1 and 2; the structures shown in the compounds 1 and 2 are as follows:
another embodiment of the present invention provides a crystalline form of a mixed source terpenoid 1, characterized in that the crystal data of the crystalline form of compound 1 is: orthorhombic, space group P2 12121Cell parameter of
α＝90°,β＝90°,γ＝90°,
Z＝4,Dx＝1.260mg/mm3,μ(Cu Kα)＝0.816mm-1F (000) ═ 1272,5654 observable points [ I>2σ(I)]The final off-factor R of 0.0373, wR of 0.0902, and the Flack constant of-0.03 (6) can be observed; compound 1 has the structure
Another embodiment of the invention provides a mixed source terpenoid 2Characterized in that the crystal data for the crystalline form of compound 2 is: orthorhombic, space group P2 12121Cell parameter of
α＝90°,β＝90°,γ＝90°,
Z＝4,Dx＝1.399mg/mm3,μ(Cu Kα)＝0.892mm-1F (000) ═ 1000,4005 observable points [ I>2σ(I)]The final off-factor R of 0.0340, wR of 0.0877, and the Flack constant of 0.10(7) can be observed; compound 2 has the structure
In another embodiment of the present invention, a method for simultaneously preparing compounds 1 and 2 by using mangrove cuspidate and sea lotus endophytic fungus TGM112 is provided, which is characterized by comprising the following steps:
(1) preparing a seed culture medium, inoculating the TGM112 strain into the seed culture medium, and culturing at 26-28 ℃ for 3-4 days to obtain a seed culture solution;
(2) inoculating the seed culture solution obtained in the step (1) into a fermentation culture medium, and performing static culture at 26-28 ℃ for 21-24 days to obtain a fermented product;
(3) separating the fermentation liquor and the thalli in the fermentation product obtained in the step (2), extracting the fermentation liquor and the thalli for 3-5 times by using equal volume of ethyl acetate respectively, combining the extraction liquids, and concentrating under reduced pressure to obtain an extract;
(4) subjecting the extract obtained in step (3) to reduced pressure silica gel column chromatography, and gradient eluting with petroleum ether-ethyl acetate at ratio of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectivelyCollecting two column volumes in a gradient way, wherein the fraction obtained by gradient elution at the ratio of 30:70 is concentrated and then is subjected to Sephadex LH-20 gel column chromatography, and the eluent is chloroform: methanol at ratio of 1: 1, and subjecting to HPLC with Agilent C18,9.4 × 250mm, 7 μm, flow rate of 2mL/min, and mobile phase CH3CN:H2O45: 55 to give compound 1; wherein the fraction obtained by gradient of 10:90 is concentrated and then is subjected to Sephadex LH-20 gel column chromatography, and the eluent is chloroform: methanol (1: 1), and High Performance Liquid Chromatography (HPLC) with Agilent C18,9.4 × 250mm, 7 μm, flow rate of 2mL/min, and mobile phase of MeOH/H2O45: 55, finally compound 2 is obtained.
Wherein the ratio of the eluent or the mobile phase is volume ratio; the seed culture medium contains 1.5-3.0% of glucose, 0.1-0.5% of yeast extract, 0.1-0.5% of peptone, 0.11-0.6% of crude sea salt and a proper amount of water; the fermentation culture medium contains 1.6-3.5% of glucose, 0.1-0.5% of yeast extract, 0.1-0.5% of peptone, 0.11-0.6% of crude sea salt and a proper amount of water; the percentages are weight percentages; the seed culture medium and the fermentation culture medium are both sterilized at 120 ℃ for 25-30 minutes.
Another embodiment of the present invention provides a process for preparing a crystalline form of compound 1 or 2, characterized by comprising the steps of: dissolving the compound 1 or 2 in an organic solvent, standing for natural crystallization, and obtaining the crystal form of the compound 1 or 2 after 2-7 days. The organic solvent is preferably one or more of methanol, ethanol, ethyl acetate and acetone.
Another embodiment of the present invention provides the use of mangrove cuspidate hydatid endophytic fungus TGM112 in the preparation of the crystalline form of compound 1 or 2.
The invention provides a pharmaceutical composition, which is characterized in that the crystal forms of the compounds 1 and 2 or the pharmaceutically acceptable salts thereof are used as active ingredients.
The pharmaceutical composition provided by the invention can also contain other insecticidal drugs; pharmaceutically acceptable adjuvants (preferably pharmaceutically acceptable carriers, diluents or excipients) may also be included. The dosage form of the pharmaceutical composition can be solid preparation, semi-solid preparation or liquid preparation.
Another embodiment of the present invention provides the use of a crystalline form of compound 1,2 or a pharmaceutically acceptable salt thereof in the preparation of a pesticide. In particular to the application in the preparation of the drug for killing cotton bollworms and nematodes.
The term "pharmaceutically acceptable salts" as used herein refers to non-toxic inorganic or organic acid and/or base addition salts, as described in "Salt selection for basic drugs", int.J.pharm. (1986),33, 201-217.
Drawings
FIG. 1 is a X-Ray diagram of Compound 1;
FIG. 2 is an ECD spectrum of Compound 1;
FIG. 3 is a graph of X-Ray for Compound 2;
figure 4 is the ECD spectrum of compound 2.
Detailed Description
In order to facilitate a further understanding of the invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the present invention and are not intended to limit the scope or the principle of the present invention, and the embodiments of the present invention are not limited to the following.
Example 1
(1) Strain culture of TGM112
Preparing a seed culture medium: 80g of glucose, 8g of peptone, 8g of yeast extract, 10g of crude sea salt and 4.0L of water are averagely distributed in 8 conical flasks with 1000mL and are sterilized at 120 ℃ for 25-30 minutes.
Inoculating the TGM112 strain into a prepared seed culture medium, and culturing for 3 days at 26-28 ℃ to obtain a seed culture solution;
(2) fermentation of TGM112
Preparing a fermentation medium: 1.1kg of glucose, 100g of peptone, 100g of yeast extract, 125g of sea salt and 50L of water are averagely distributed in 75 conical flasks with 1000mL and are sterilized for 25-30 minutes at 120 ℃.
And (2) taking a proper amount of the seed culture solution obtained in the step (1) to be inoculated into a conical flask filled with a fermentation culture medium, and performing static culture at 26-28 ℃ for 21 days to obtain a fermented product.
(3) Preparation of extract
Separating the fermentation liquor and the thalli in the fermentation product obtained in the step (2), extracting the fermentation liquor and the thalli for 3 times by using equal volume of ethyl acetate respectively, combining the extraction liquids, and concentrating under reduced pressure to obtain an extract;
(4) isolation of Compound 1-2
And (3) performing reduced pressure silica gel column chromatography on the extract obtained in the step (3), performing gradient elution by adopting petroleum ether-ethyl acetate according to the ratio of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100, collecting two column volumes in each gradient, concentrating the fraction obtained by gradient elution of 30:70, performing Sephadex LH-20 gel column chromatography, and eluting by using chloroform: methanol at ratio of 1: 1, and subjecting to HPLC with Agilent C18,9.4 × 250mm, 7 μm, flow rate of 2mL/min, and mobile phase CH3CN:H2O45: 55 to give compound 1(16.2 mg); wherein the fraction obtained by gradient of 10:90 is concentrated and then is subjected to Sephadex LH-20 gel column chromatography, and the eluent is chloroform: methanol (1: 1), and High Performance Liquid Chromatography (HPLC) with Agilent C18,9.4 × 250mm, 7 μm, flow rate of 2mL/min, and mobile phase of MeOH/H2O45: 55, to give compound 2(13.3 mg).
Of compounds 1 and 21H and13C-NMR(400/100MHz,aCDCl3.bCD3OD) data are as in table 1 below.
TABLE 1 of Compounds 1 and 21H and13C-NMR(400/100MHz,aCDCl3.bCD3OD) data (ppm)
From1H and13the C NMR spectrum of compound 1 was observed to have 6 carbonyl carbons and 4 olefinic carbons, which presumably present a hexacyclic ring system with the basic backbone of the mixed source terpene. In addition to this, 1 alkene hydrogen signal appears in the low field regionH6.78(q, J ═ 5.6Hz, H-20) and 2 terminal double bond hydrogen signalsH5.83(dd, J ═ 24.0,1.6Hz, H-13), 3 vicinal oxymethylene hydrogen signalsH5.93(m, H-11),5.47(dd, J ═ 10.0,5.6Hz, H-7), and 5.19(q, J ═ 6.8Hz, H-5'), 3 methylene signalsH2.61(m, H-2),2.53(m, H-1a) and 2.33(m, H-1b),1.86(d, J ═ 5.2Hz, H-6a) and 1.83(d, J ═ 5.2Hz, H-6b), the above hydrogen and carbon spectra information suggests that this compound is very similar to the known compound 1, 2-hydro-terredhydroaustin, except that there are two fewer olefinic carbon signals in compound 1 (only two less olefinic carbon signals are present: (b, H-1 b))C116.7and 137.0) plus one carbonyl signal (a)C191.7), it is speculated that the carbon-carbon double bonds of C-1 ' and C-2 ' in the known compounds are oxidized to the carbonyl group of C-2 ' in compound 1, as evidenced by the high resolution data. The attachment position of the C-9 ' methyl group is determined by HMBC, where 9' -Me is associated with C-2 '/C-3 '/C-11, suggesting that the methyl group is attached to C-3', and the 2-methylcrotonate unit is also determined by HMBC attachment, where H-22 is associated with C-18/C-19/C-20. Based on the above inference that the planar structure of compound 1 was determined, the relative configuration of compound 1 was determined by NOESY, where it can be seen in the NOESY spectra that H-11 is associated with 15-Me and H-7, H-7 is associated with 10'-Me, and 9' -Me is associated with H-5 'and 12-Me, i.e., 9' -Me, H-5 'and 12-Me are in the α -plane and H-7, H-11,15-Me and 10' -Me are in the β -plane. The absolute configuration of compound 1 was determined by X-Ray diffraction and ECD calculations, i.e. the absolute configuration of compound was 5S,7R,8S,9R,11S,3 'R, 5' R,6'R,7' S. Named as peniiansitinoid A.
Determination of the structure of compound 2: from high resolution Mass Spectrometry data 473.1811[ M + H]+The molecular formula of the compound 2 is calculated to be C25H28O9The degree of unsaturation being12。
1H NMR and13the C-NMR spectrum is very similar to that of the known compound dehydroaustinol, in which C-7 is methylene [ 2 ], except for the difference in C-7 chemical shiftH1.70(m) andC27.7(CH2)]wherein C-7 in the compound 2 is vicinal oxymethylene [ 2 ]H4.32(dd, J ═ 12.0,4.8Hz) andC64.7(CH)]h-7 is also seen to correlate with C-5/C-8/C-9 in HMBC. The absolute configuration of the compound 2 is determined by NOESY, X-Ray and ECD calculation, namely the absolute configuration of the compound 2 is 5S,7R,8S,9R,11R,3'S,5' R,6'R,7' S and is named penicianstinidB.
Example 2
Dissolving 2.0mg of compound 1 in 2mL of acetone, standing for natural crystallization, and obtaining colorless crystals after 3 days, wherein the crystal structure of the colorless crystals adopts CuK alpha rays by a Gemini super diffractometer (Xcalibur, Atlas, Gemini ultra diffractometer)
Diffraction data was collected by scanning at 120.01(10) K.
Crystal data for compound 1: orthorhombic, space group P2 12121Cell parameter of
α＝90°,β＝90°,γ＝90°,
Z＝4,Dx＝1.260mg/mm3,μ(Cu Kα)＝0.816mm-1F (000) ═ 1272,5654 observable points [ I>2σ(I)]The final off-factor R of 0.0373, wR of 0.0902 and a Flack constant of-0.03 (6) can be observed.
Dissolving 2.0mg of compound 2 in 2mL of methanol, standing for natural crystallization, and obtaining colorless crystals after 4 days, wherein the crystal structure of the colorless crystals adopts CuK alpha rays by a Gemini super diffractometer (Xcalibur, Atlas, Gemini ultra diffractometer)
Diffraction data was collected by scanning at 120.01(10) K.
Crystal data for compound 2: orthorhombic, space group P2 12121Cell parameter of
α＝90°,β＝90°,γ＝90°,
Z＝4,Dx＝1.399mg/mm3,μ(Cu Kα)＝0.892mm-1F (000) ═ 1000,4005 observable points [ I>2σ(I)]The final off-factor R of 0.0340, wR of 0.0877, and the Flack constant of 0.10(7) can be observed.
Example 3
(1) Strain culture of TGM112
Seed medium (10.0L) was prepared: 1.5% of glucose (weight percentage, the same below), 0.5% of yeast extract, 0.1% of peptone, 0.11% of crude sea salt and the balance of water; the mixture is averagely distributed into 16 conical flasks with 1000mL, and is sterilized at 120 ℃ for 25-30 minutes.
Inoculating the TGM112 strain into a prepared seed culture medium, and culturing for 4 days at 26-28 ℃ to obtain a seed culture solution;
(2) fermentation of TGM112
Preparing a fermentation medium (100L): 1.6% of glucose (weight percentage, the same below), 0.5% of yeast extract, 0.1% of peptone, 0.11% of crude sea salt and the balance of water; the mixture is averagely distributed into 150 conical flasks with the volume of 1000mL and is sterilized at 120 ℃ for 25-30 minutes.
And (2) taking a proper amount of the seed culture solution obtained in the step (1) to be inoculated into a conical flask filled with a fermentation culture medium, and performing static culture at 26-28 ℃ for 24 days to obtain a fermented product.
(3) Preparation of extract
Separating the fermentation liquor and the thalli in the fermentation product obtained in the step (2), extracting the fermentation liquor and the thalli for 5 times by using equal volume of ethyl acetate respectively, combining the extraction liquids, and concentrating under reduced pressure to obtain an extract;
(4) isolation of Compound 1-2
And (3) performing reduced pressure silica gel column chromatography on the extract obtained in the step (3), performing gradient elution by adopting petroleum ether-ethyl acetate according to the ratio of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100, collecting two column volumes in each gradient, concentrating the fraction obtained by gradient elution of 30:70, performing Sephadex LH-20 gel column chromatography, and eluting by using chloroform: methanol at ratio of 1: 1, and subjecting to HPLC with Agilent C18,9.4 × 250mm, 7 μm, flow rate of 2mL/min, and mobile phase CH3CN:H2O45: 55 to give compound 1(32.5 mg); wherein the fraction obtained by gradient of 10:90 is concentrated and then is subjected to Sephadex LH-20 gel column chromatography, and the eluent is chloroform: methanol (1: 1), and High Performance Liquid Chromatography (HPLC) with Agilent C18,9.4 × 250mm, 7 μm, flow rate of 2mL/min, and mobile phase of MeOH/H2O45: 55, to give compound 2(25.4 mg).
Example 4
(1) Strain culture of TGM112
Seed medium (1.0L) was prepared: 3.0 percent of glucose (weight percentage, the same below), 0.1 percent of yeast extract, 0.5 percent of peptone, 0.6 percent of crude sea salt and the balance of water; the mixture is evenly distributed into 3 500mL conical bottles and is sterilized for 25 to 30 minutes at 120 ℃.
Inoculating the TGM112 strain into a prepared seed culture medium, and culturing for 4 days at 26-28 ℃ to obtain a seed culture solution;
(2) fermentation of TGM112
Preparing a fermentation medium (10L): 3.5 percent of glucose (weight percentage, the same below), 0.1 percent of yeast extract, 0.5 percent of peptone, 0.6 percent of crude sea salt and the balance of water; the mixture is evenly distributed into 15 conical flasks of 1000mL and is sterilized at 120 ℃ for 25-30 minutes.
And (2) taking a proper amount of the seed culture solution obtained in the step (1) to be inoculated into a conical flask filled with a fermentation culture medium, and performing static culture at 26-28 ℃ for 22 days to obtain a fermented product.
(3) Preparation of extract
Separating the fermentation liquor and the thalli in the fermentation product obtained in the step (2), extracting the fermentation liquor and the thalli for 5 times by using equal volume of ethyl acetate respectively, combining the extraction liquids, and concentrating under reduced pressure to obtain an extract;
(4) isolation of Compound 1-2
And (3) performing reduced pressure silica gel column chromatography on the extract obtained in the step (3), performing gradient elution by adopting petroleum ether-ethyl acetate according to the ratio of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100, collecting two column volumes in each gradient, concentrating the fraction obtained by gradient elution of 30:70, performing Sephadex LH-20 gel column chromatography, and eluting by using chloroform: methanol at ratio of 1: 1, and subjecting to HPLC with Agilent C18,9.4 × 250mm, 7 μm, flow rate of 2mL/min, and mobile phase CH3CN:H2O45: 55 to give compound 1; wherein the fraction obtained by gradient of 10:90 is concentrated and then is subjected to Sephadex LH-20 gel column chromatography, and the eluent is chloroform: methanol (1: 1), and High Performance Liquid Chromatography (HPLC) with Agilent C18,9.4 × 250mm, 7 μm, flow rate of 2mL/min, and mobile phase of MeOH/H2O45: 55, finally compound 2 is obtained.
EXAMPLE 5 determination of the anti-insect Activity of Compounds 1,2 of the present invention
The test method comprises the following steps: the activity of each pair of bollworm larvae and nematodes of the compounds 1 and 2 was tested.
Cotton bollworm activity assay, equal amounts of artificial feed mixed with samples of different concentrations (200,100,50,25and 12.5. mu.L/well) were added to 6-well plates in triplicate. A positive group, a blank group and a negative group are set, corresponding artificial feed and positive drugs (200,100,50,25and12.5 mu L/hole) are respectively added into the positive control group, the blank control is equivalent artificial feed, and the negative control is DMSO (200,100,50,25and12.5 mu L/hole) with different concentrations. And (3) placing the 6-hole plate at room temperature for continuous culture for one week, observing the growth condition of the cotton bollworm larvae, and observing and recording the growth and death conditions of the cotton bollworms every 2 days. Azadirachtin (azadirachtin) was used as a positive control.
Nematode activity assay, 40. mu.L of E.coli OP50, 5. mu.L of L1 old nematodes (30-40 strips) in M9 medium, 1.2. mu.L of chloramphenicol, all compounds added at 0,1,5,10,20, 40. mu.g/mL, respectively, were added to a 48-well plate, and all well volumes were filled to 400. mu.L with medium, and three sets of replicates were set. A positive group, a negative group and a blank group are set, wherein the positive group is added with a medium and a positive drug (0,1,5,10,20,40 mu g/mL), the negative group is added with only DMSO (0,1,5,10,20,40 mu g/mL), and the blank group is not added with anything. The cells were incubated at 24 ℃ for 60 hours in the dark and observed every twelve hours, and the growth and death of the nematodes, levamisole (levamisole), were recorded.
The test results are as follows:
table 2:
in the above table: IC (integrated circuit)50: half inhibitory concentration (half inhibitory concentration).
EC50: half effective concentration (concentration for 50% of maximum effect).
AzadirachtinaIs a positive control of the cotton bollworm group.
LevamisolebIs a positive control of the nematode group
The test result shows that the two compounds have certain anti-insect activity, and the compound 1 and the compound 2 have inhibitory activity and IC (Integrated Circuit) on H50The values were all 200. mu.g/mL. The compound 1 and the compound 2 have better lethal activity, EC, on C50The values were all 10. mu.g/mL.
Claims (10)
1. A crystalline form of a mixed source terpenoid 1, characterized in that the crystal data of the crystalline form of Compound 1 is: orthorhombic, space group P212121Cell parameter of
α＝90°,β＝90°,γ＝90°,
Z＝4,Dx＝1.260mg/mm3,μ(Cu Kα)＝0.816mm-1F (000) ═ 1272,5654 observable points [ I>2σ(I)]The final off-factor R of 0.0373, wR of 0.0902, and the Flack constant of-0.03 (6) can be observed; compound 1 has the structure
2. A process for preparing a crystalline form of compound 1 according to claim 1, characterized by the steps of: dissolving the compound 1 in an organic solvent, standing for natural crystallization, and obtaining the crystal form of the compound 1 after 2-7 days.
3. The method of claim 2, wherein the organic solvent is selected from one or more of methanol, ethanol, ethyl acetate, and acetone.
4. A pharmaceutical composition characterized by a crystalline form of compound 1 or a pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient.
5. The pharmaceutical composition of claim 4, further comprising an additional pesticidal agent.
6. The pharmaceutical composition according to any one of claims 4 to 5, further comprising a pharmaceutically acceptable excipient.
7. The pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable excipient is selected from the group consisting of a pharmaceutically acceptable carrier, diluent, or excipient.
8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is in a solid, semi-solid, or liquid form.
9. Use of a crystalline form of compound 1 as defined in claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of a pesticide.
10. The use of a crystalline form of compound 1 as claimed in claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a bollworm and nematode killing medicament.
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| CN202010874641.5A CN111909858B (en) | 2018-12-04 | 2018-12-04 | Mangrove endophytic fungus TGM112 secondary metabolite and application thereof |
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| CN109486685A (en) | 2019-03-19 |
| CN111892611B (en) | 2022-12-20 |
| CN111909858A (en) | 2020-11-10 |
| CN111892611A (en) | 2020-11-06 |
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